Transition metal ions play highly specialized roles in biological catalysis and all cells encode regulatory machinery that controls their intracellular availability so that mis-metallation and metal toxicity is avoided. In bacteria, this process is transcriptionally regulated by a panel of metal sensor proteins in response to changes in metal bioavailability. The proposed experiments strongly integrate structural and cell biological approaches to elucidate determinants of metal homeostasis and cross-talk in the respiratory pathogen Streptococcus pneumoniae, focusing on zinc, manganese and copper, three metals whose concentrations change dramatically during the course of invasive disease. In the next project period, our specific aims are to 1) Obtain a structural and physicochemical characterization of the novel zinc uptake regulator S. pneumoniae AdcR (adhesin competence repressor). Here we propose NMR, structural and thermodynamic studies of wild-type and mutant AdcRs in an effort to understand allosteric activation of DNA operator binding; 2) Perform a direct test of the thermodynamic set-point model for zinc homeostasis in S. pneumoniae, which posits that free zinc is buffered in a concentration range dictated by the zinc affinities of the uptake (AdcR) and efflux (SczA, streptococcal czcD activator) regulators. Since intracellular metallation status is dictated by free or chelatable zinc, not total zinc, we propose to measure and manipulate free Zn in the cytoplasm using a FRET-based imaging assay; 3) Obtain structural and mechanistic insights into copper resistance and trafficking in S. pneumoniae, a process characterized by features not previously observed; and 4) Carry out structural studies of the CuI sensor CsoR and CsoR-related proteins in Mycobacterium tuberculosis and other bacilli. Small angle X-ray scattering, NMR and crystallographic studies are proposed. Since an emerging aspect of nutritional immunity to infection by bacterial human pathogens is the host control of metal availability, these studies lay the foundation for our long-term goal of disrupting metal availability or inducing metal toxicity by developing new antimicrobial agents that selectively target key components of transition metal homeostasis systems.